Revisiting the Elusive Hepatitis C Vaccine.

The impactful discovery and subsequent characterisation of hepatitis C virus (HCV), an RNA virus of the flavivirus family, led to the awarding of the 2020 Nobel Prize in Physiology or Medicine to Harvey J. Alter, Michael Houghton and Charles M. Rice [...].

T Cell Memory to Vaccination.

Most immune responses associated with vaccination are controlled by specific T cells of a CD4+ helper phenotype which mediate the generation of effector antibodies, cytotoxic T lymphocytes (CTLs), or the activation of innate immune effector cells. A rapidly growing understanding of the generation, maintenance, activity, and measurement of such T cells is leading to vaccination strategies with greater efficacy and potentially greater microbial coverage.

Anti-bacterial antibody and T cell responses in bronchiectasis are differentially associated with lung colonization and disease.

BACKGROUND: As a way to determine markers of infection or disease informing disease management, and to reveal disease-associated immune mechanisms, this study sought to measure antibody and T cell responses against key lung pathogens and to relate these to patients' microbial colonization status, exacerbation history and lung function, in Bronchiectasis (BR) and Chronic Obstructive Pulmonary Disease (COPD). METHODS: One hundred nineteen patients with stable BR, 58 with COPD and 28 healthy volunteers were recruited and spirometry was performed. Bacterial lysates were used to measure specific antibody responses by ELISA and T cells by ELIspot. Cytokine secretion by lysate-stimulated T cells was measured by multiplex cytokine assay whilst activation phenotype was measured by flow cytometry. RESULTS: Typical colonization profiles were observed in BR and COPD, dominated by P.aeruginosa, H.influenzae, S.pneumoniae and M.catarrhalis. Colonization frequency was greater in BR, showing association with increased antibody responses against P.aeruginosa compared to COPD and HV, and with sensitivity of 73% and specificity of 95%. Interferon-gamma T cell responses against P.aeruginosa and S.pneumoniae were reduced in BR and COPD, whilst reactive T cells in BR had similar markers of homing and senescence compared to healthy volunteers. Exacerbation frequency in BR was associated with increased antibodies against P. aeruginosa, M.catarrhalis and S.maltophilia. T cell responses against H.influenzae showed positive correlation with FEV1% (r = 0.201, p = 0.033) and negative correlation with Bronchiectasis Severity Index (r = - 0.287, p = 0.0035). CONCLUSION: Our findings suggest a difference in antibody and T cell immunity in BR, with antibody being a marker of exposure and disease in BR for P.aeruginosa, M.catarrhalis and H.influenzae, and T cells a marker of reduced disease for H.influenzae.

An adjuvant-modulated vaccine response in human whole blood.

The restimulation of an immune memory response by in vitro culture of blood cells with a specific antigen has been used as a way to gauge immunity to vaccines for decades. In this commentary we discuss a less appreciated application to support vaccine process development. We report that human whole blood from pre-primed subjects can generate a profound adjuvant-modulated, antigen-specific response to several different vaccine formulations. The response is able to differentiate subtle changes in the quality of an immune memory response to vaccine formulations and can be used to select optimal conditions relating to a particular manufacture process step. While questions relating to closeness to in vivo vaccination remain, the approach is another big step nearer to the more relevant human response. It has special importance for new adjuvant development, complementing other preclinical in vivo and in vitro approaches to considerably de-risk progression of novel vaccines before and throughout early clinical development. Broader implications of the approach are discussed.

Leptin and Pro-Inflammatory Stimuli Synergistically Upregulate MMP-1 and MMP-3 Secretion in Human Gingival Fibroblasts.

INTRODUCTION: Gingival fibroblast-mediated extracellular matrix remodelling is implicated in the pathogenesis of periodontitis, yet the stimuli that regulate this response are not fully understood. The immunoregulatory adipokine leptin is detectable in the gingiva, human gingival fibroblasts express functional leptin receptor mRNA and leptin is known to regulate extracellular matrix remodelling responses in cardiac fibroblasts. We therefore hypothesised that leptin would enhance matrix metalloproteinase secretion in human gingival fibroblasts. METHODS AND RESULTS: We used in vitro cell culture to investigate leptin signalling and the effect of leptin on mRNA and protein expression in human gingival fibroblasts. We confirmed human gingival fibroblasts expressed cell surface leptin receptor, found leptin increased matrix metalloproteinase-1, -3, -8 and -14 expression in human gingival fibroblasts compared to unstimulated cells, and observed that leptin stimulation activated MAPK, STAT1/3 and Akt signalling in human gingival fibroblasts. Furthermore, leptin synergised with IL-1 or the TLR2 agonist pam2CSK4 to markedly enhance matrix metalloproteinase-1 and -3 production by human gingival fibroblasts. Signalling pathway inhibition demonstrated ERK was required for leptin-stimulated matrix metalloproteinase-1 expression in human gingival fibroblasts; whilst ERK, JNK, p38 and STAT3 were required for leptin+IL-1- and leptin+pam2CSK4-induced matrix metalloproteinase-1 expression. A genome-wide expression array and gene ontology analysis confirmed genes differentially expressed in leptin+IL-1-stimulated human gingival fibroblasts (compared to unstimulated cells) were enriched for extracellular matrix organisation and disassembly, and revealed that matrix metalloproteinase-8 and -12 were also synergistically upregulated by leptin+IL-1 in human gingival fibroblasts. CONCLUSIONS: We conclude that leptin selectively enhances the expression and secretion of certain matrix metalloproteinases in human gingival fibroblasts, and suggest that gingival fibroblasts may have an ECM-degrading phenotype during conditions of hyperleptinaemia (e.g., obesity, type 2 diabetes mellitus, exogenous leptin therapy).

Antibody and T-cell responses associated with experimental human malaria infection or vaccination show limited relationships.

This study examined specific antibody and T-cell responses associated with experimental malaria infection or malaria vaccination, in malaria-naive human volunteers within phase I/IIa vaccine trials, with a view to investigating inter-relationships between these types of response. Malaria infection was via five bites of Plasmodium falciparum-infected mosquitoes, with individuals reaching patent infection by 11-12 days, having harboured four or five blood-stage cycles before drug clearance. Infection elicited a robust antibody response against merozoite surface protein-119 , correlating with parasite load. Classical class switching was seen from an early IgM to an IgG1-dominant response of increasing affinity. Malaria-specific T-cell responses were detected in the form of interferon-γ and interleukin-4 (IL-4) ELIspot, but their magnitude did not correlate with the magnitude of antibody or its avidity, or with parasite load. Different individuals who were immunized with a virosome vaccine comprising influenza antigens combined with P. falciparum antigens, demonstrated pre-existing interferon-γ, IL-2 and IL-5 ELIspot responses against the influenza antigens, and showed boosting of anti-influenza T-cell responses only for IL-5. The large IgG1-dominated anti-parasite responses showed limited correlation with T-cell responses for magnitude or avidity, both parameters being only negatively correlated for IL-5 secretion versus anti-apical membrane antigen-1 antibody titres. Overall, these findings suggest that cognate T-cell responses across a range of magnitudes contribute towards driving potentially effective antibody responses in infection-induced and vaccine-induced immunity against malaria, and their existence during immunization is beneficial, but magnitudes are mostly not inter-related.

Screening vaccine formulations for biological activity using fresh human whole blood.

Understanding the relevant biological activity of any pharmaceutical formulation destined for human use is crucial. For vaccine-based formulations, activity must reflect the expected immune response, while for non-vaccine therapeutic agents, such as monoclonal antibodies, a lack of immune response to the formulation is desired. During early formulation development, various biochemical and biophysical characteristics can be monitored in a high-throughput screening (HTS) format. However, it remains impractical and arguably unethical to screen samples in this way for immunological functionality in animal models. Furthermore, data for immunological functionality lag formulation design by months, making it cumbersome to relate back to formulations in real-time. It is also likely that animal testing may not accurately reflect the response in humans. For a more effective formulation screen, a human whole blood (hWB) approach can be used to assess immunological functionality. The functional activity relates directly to the human immune response to a complete formulation (adjuvant/antigen) and includes adjuvant response, antigen response, adjuvant-modulated antigen response, stability, and potentially safety. The following commentary discusses the hWB approach as a valuable new tool to de-risk manufacture, formulation design, and clinical progression.

How do malaria parasites activate dendritic cells?

Evaluation of: Wu X, Gowda NM, Kumar S, Gowda S: Protein-DNA complex is the exclusive malaria parasite component that activates dendritic cells and triggers innate immune responses. J. Immunol. 184(8), 4338-4348 (2010). Malaria parasites induce strong proinflammatory immune responses upon infection. These responses, driven largely by CD4+ Th1 cells, help the body to control malaria parasitemia. When excessive, inflammatory responses contribute to the pathology observed in malaria infection. Dendritic cells (DCs) are innate immune cells that activate Th1 cells in malaria infection via the secretion of the cytokine IL-12. It remains unclear precisely which components of malaria-infected red blood cells are responsible for activating DCs. In this study, Wu et al. set out to deconstruct malaria-infected red blood cells to determine the immunogenic components that induce production of the proinflammatory cytokines IL-12 and TNF-alpha from DCs. The authors suggest that parasite DNA complexed with protein is the main trigger for activation of DCs in malaria-infected red blood cells.

Comparing human T cell and NK cell responses in viral-based malaria vaccine trials.

Vaccination with viral-based vaccines continues to hold promise for the prevention of malaria. Whilst antigen-specific T cell responses are considered a major aim of such an approach, a role for induced NK cells as anti-malarial effector cells, or in shaping T cell responses, has received less attention. In this study naïve human volunteers were vaccinated in a prime-boost vaccination regimen comprising recombinant viral vectors fowlpox (FP9) and modified vaccinia Ankara (MVA) encoding liver-stage antigens, or a virosome vaccine. Significant T cell responses specific for the vectored vaccine antigens were demonstrated by IFNgamma ELISPOT and intracellular cytokine staining (ICS) for IFNgamma and IL-2, the ICS being associated with increased time to parasitaemia following subsequent challenge. Numbers of CD56(bright) lymphocytes increased significantly following vaccination, as did CD3(+) CD56(+) lymphocytes, whilst CD56(dim) cells did not. No such increases were seen with the virosome vaccine. There was no significant correlation of these CD56(+) populations with the antigen-specific T cell responses nor time to parasitaemia. To investigate pathways of immune activation that could contribute to these lymphocyte responses, viral vectors were shown in vitro to efficiently infect APCs but not lymphocytes, and stimulated inflammatory cytokines such as type I interferons. In conclusion, measuring antigen-specific T cells is more meaningful than NK cells in these vaccination regimens.

Multiple functions of human T cells generated by experimental malaria challenge.

Protective immunity generated following malaria infection may be comprised of Ab or T cells against malaria Ag of different stages; however, the short-lived immunity that is observed suggests deficiency in immune memory or regulatory activity. In this study, cellular immune responses were investigated in individuals receiving Plasmodium falciparum sporozoite challenge by the natural (mosquito bite) route as part of a malaria vaccine efficacy trial. Parasitemia, monitored by blood film microscopy and PCR, was subsequently cleared with drugs. All individuals demonstrated stable IFN-gamma, IL-2 and IL-4 ex vivo ELISPOT effector responses against P. falciparum-infected RBC (iRBC) Ag, 28 and 90 days after challenge. However, infected RBC-specific central memory responses, as measured by IFN-gamma cultured ELISPOT, were low and unstable over time, despite CD4(+) T cells being highly proliferative by CFSE dilution, and showed an inverse relationship to parasite density. In support of the observation of poor memory, co-culture experiments showed reduced responses to common recall Ag, indicating malaria-specific regulatory activity. This activity could not be accounted for by the expression of IL-10, TGF-beta, FOXP3 or CTLA-4, but proliferating T cells expressed high levels of CD95, indicating a pro-apoptotic phenotype. Lastly, there was an inverse relationship between FOXP3 expression, when measured 10 days after challenge, and ex vivo IFN-gamma measured more than 100 days later. This study shows that malaria infection elicits specific Th1 and Th2 effector cells, but concomitant weak central memory and regulatory activity, which may help to explain the short-lived immunity observed.

The relationship between human effector and memory T cells measured by ex vivo and cultured ELISPOT following recent and distal priming.

Maintenance of T-cell responses is an essential feature in protection from many infectious diseases that must be harnessed in vaccination. The relationship between effector T-cell responses and more durable and highly proliferative T-cell memory, particularly in humans, is not well understood. In this study, effector T-cell responses were measured by overnight ex vivo interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot-forming cell assay (ELISPOT), whereas memory T cells were measured by 10-day culture followed by IFN-gamma ELISPOT (cultured ELISPOT). We observed a significant correlation between IFN-gamma responses to CD4-stimulatory, but not to CD8-stimulatory, recall antigens measured by these assays, suggesting a divergence in regulation. In vaccine trial participants who received a prime-boost vaccination regimen comprising malaria antigens delivered by poxviruses, there was a correlation between ex vivo and cultured responses on day 7, but not 3 months post-vaccination, with the ratio of cultured : ex vivo response increasing over time. To compare responses revealed by cultured ELISPOT in more detail, tetramers comprising viral recall antigens were used to ascribe effector-memory and central-memory T-cell phenotypes through CCR7 and CD62L costaining. For CD8(+) responses the effector phenotype decreased during the initial culture period and memory populations remained high within the resulting 20-fold to 50-fold increased IFN-gamma-secreting or tetramer(+) population. This was less marked for CD4(+) responses, which had higher starting memory phenotype. Depletion of these central-memory T-cell populations generally ablated responses in cultured ELISPOT and reduced ex vivo responses. This study highlights differences between CD4(+) and CD8(+) effector and memory T cells, and the more complex phenotype of CD4(+) T cells.

A prostate cancer vaccine comprising whole cells secreting IL-7, effective against subcutaneous challenge, requires local GM-CSF for intra-prostatic efficacy.

A panel of cytokine-secreting RM-9 prostate cancer cells were tested as whole cell vaccines to determine their capacity to evoke an anti-prostate cancer immune response. In our model, vaccines secreting mGM-CSF or mIL-7 resulted in the highest increase in circulating T lymphocytes after vaccination, prolonged survival and, in a proportion of animals, tumor-free survival. Anti-tumor effects were more evident after a subcutaneous RM-9 challenge than after an intraprostatic challenge. However, when the RM-9/mGM-CSF cell line was used as intraprostatic tumor challenge, protection after RM-9/mIL-7 vaccination was restored.

Dendritic cells in Plasmodium infection.

Infection with Plasmodium parasites (malaria) contributes greatly to morbidity and mortality in affected areas. Interaction of the protozoan with the immune system has a critical role in the pathogenesis of the disease, but may also hold a key to containing parasite numbers through specific immune responses, which vaccine development aims to harness. A central player in the generation of such immune responses is the dendritic cell. However, Plasmodium parasites appear to have profound activating and suppressing effects on dendritic cell function, which may enhance immunopathology or facilitate the parasite's survival by depressing beneficial immunity. Furthermore, immune responses to other infections and vaccines may be impaired. A greater understanding of the effects of the parasite on dendritic cells will contribute to insight and potential defeat of this infectious disease.

Correlation of memory T cell responses against TRAP with protection from clinical malaria, and CD4 CD25 high T cells with susceptibility in Kenyans.

BACKGROUND: Immunity to malaria develops naturally in endemic regions, but the protective immune mechanisms are poorly understood. Many vaccination strategies aim to induce T cells against diverse pre-erythrocytic antigens, but correlates of protection in the field have been limited. The objective of this study was to investigate cell-mediated immune correlates of protection in natural malaria. Memory T cells reactive against thrombospondin-related adhesive protein (TRAP) and circumsporozoite (CS) protein, major vaccine candidate antigens, were measured, as were frequencies of CD4(+) CD25(high) T cells, which may suppress immunity, and CD56(+) NK cells and gammadelta T cells, which may be effectors or may modulate immunity. METHODOLOGY AND PRINCIPAL FINDINGS: 112 healthy volunteers living in rural Kenya were entered in the study. Memory T cells reactive against TRAP and CS were measured using a cultured IFNgamma ELISPOT approach, whilst CD4(+) CD25(high) T cells, CD56(+) NK cells, and gammadelta T cells were measured by flow cytometry. We found that T cell responses against TRAP were established early in life (<5 years) in contrast to CS, and cultured ELISPOT memory T cell responses did not correlate with ex-vivo IFNgamma ELISPOT effector responses. Data was examined for associations with risk of clinical malaria for a period of 300 days. Multivariate logistic analysis incorporating age and CS response showed that cultured memory T cell responses against TRAP were associated with a significantly reduced incidence of malaria (p = 0.028). This was not seen for CS responses. Higher numbers of CD4(+) CD25(high) T cells, potentially regulatory T cells, were associated with a significantly increased risk of clinical malaria (p = 0.039). CONCLUSIONS: These data demonstrate a role for central memory T cells in natural malarial immunity and support current vaccination strategies aimed at inducing durable protective T cell responses against the TRAP antigen. They also suggest that CD4(+) CD25(high) T cells may negatively affect naturally acquired malarial immunity.

Viral vector vaccines make memory T cells against malaria.

Vaccines that comprise attenuated viral vectors encoding antigens from target pathogens generate potent T-cell responses. One such pathogen is malaria, and in particular the liver stage of its life cycle. Immunogenicity and efficacy studies in animals and humans have revealed the generation of memory T cells of both the central and effector phenotypes, depending on the viral vectors used in the malaria vaccination regime (viral species and serotype, combination and sequence for prime-boost) and suggest a divergence in their protective role. Being able to influence the memory T-cell make-up in a rational manner may allow us to develop more efficacious vaccines.

Nitroreductase-based therapy of prostate cancer, enhanced by raising expression of heat shock protein 70, acts through increased anti-tumour immunity.

Gene-directed enzyme-prodrug therapy (GDEPT) using nitroreductase (NTR), with efficient adenoviral delivery, and CB1954 (CB), is an effective means of directly killing tumours. However, an immune-mediated bystander effect remains an important product of GDEPT since it is often critical to the elimination of untransduced tumour cells both locally and at distal metastatic sites through generation of tumour-specific immunity without the need for tumour antigen identification or the generation of a personalised vaccine. The mode of induced tumour cell death is thought to contribute to the immunisation process, together with the induction and release of stress proteins. Here, RM-9 murine prostate tumour cells were efficiently killed by adenovirally delivered NTR/CB treatment both in vitro and in vivo, and bystander effects were observed. Cells appeared to die by pathways that suggest necrosis more than that of classical apoptosis. NTR/CB-induced expression of a range of stress proteins was determined by proteomic analysis, revealing chiefly heat shock protein (HSP)25 and HSP70 upregulation, whilst immune responses in vivo were weak. In an attempt to enhance the anti-tumour effect, an adenoviral vector was constructed that co-expressed NTR and HSP70, the latter being a known immune stimulator and chaperone of antigen. This combination elicited significantly enhanced protection over NTR alone for both the treated tumour and a subsequent re-challenge. Protection was CD4+ and CD8+ T cell-dependent and was associated with tumour-specific CTL, IFNgamma and IL-5 responses. The use of such a cytotoxic and immunomodulatory gene combination in cancer therapy warrants further pursuit.